Algal polysaccharide production

ABSTRACT

Selected polysaccharides are produced from algal plant tissue by (a) separating vegetative parts or spores into (i) gametophytes, or (ii) tetrasporophytes; (b) vegetatively propagating the separated plant material; (c) harvesting the resulting plant growth; and (d) recovering the desired polysaccharides from the harvested material. Different specific polysaccharides (e.g., kappa- or lambda-carrageenan) have been found to be produced by (i) or (ii) above. This method avoids difficult post-harvest separations and is amenable to controlled vegetative propagation of the separated plant material under optimum conditions.

United States Patent [19] Chen et al.

[111 3,879,890 1 Apr. 29, 1975 ALGAL POLYSACCHARIDE PRODUCTION [75]Inventors: Lawrence C. M. Chen, Spryfield.

Nova Scotia; James S. Craigie, Halifax, Nova Scotia; Esther L.McCandless, Dundas. Ontario; Jack L. McLachlan, Halifax, Nova Scotia.all of Canada; Arthur C. Neish, deceased, late of Granville Ferry, NovaScotia, Canada, by Dorothy A. Neish, executrix; Peter F. Shacklock,Sambro; John A. Walter, Halifax, Nova Scotia, both of Canada [73]Assignee: Canadian Patents and Development Limited, Ontario, Canada 22Filed: Feb. 6, 1974 21 Appl. No.: 440,247

[52] US. Cl. 47/l.4; 260/209 R [51] Int. Cl A0lg 7/00 [58] Field ofSearch 47/l.4; 260/209 R [56] References Cited UNITED STATES PATENTS2,620,334 2/1952 Nielsen et al. 260/209 2.732.661 l/l956 Spochr etal.... 47/l.4 3.l95,27l 7/1965 Golvcke et al.. 47/l.4 3.439.449 4/1969Huff 47/l.4

Primary Examiner-Robert E. Bagwill Attorney. Agent, or Firm-Alan A.Thomson [57] ABSTRACT Selected polysaccharides are produced from algalplant tissue by (a) separating vegetative parts or spores into (i)gametophytes, or (ii) tetrasporophytes;

12 Claims, N0 Drawings ALGAL POLYSACCHARIDE PRODUCTION This invention isdirected to the production of polysaccharides from a selected alga suchthat distinct polysaccharides are produced rather than the usualmixtures. Marine algae such as Irish moss producing carrageenans are ofparticular interest.

At present the algae are harvested from natural sources without sortingthe raw material with respect to chemical constituents. Harvesting usinghand or crude methods from coastal waters and rocks is traditional andsubject to the vagaries of natural supply and seasonal labor. Thepolysaccharides are extracted from the algae with hot aqueous solutionsyielding a crude extract consisting of several polysaccharides inproportions which vary widely and unpredictably. Since these mixturesoften contain undesired components it is usually necessary to carry outchemical separation or purification steps on the product.

carrageenans, water-soluble polysaccharides of some commercialimportance, occur in quantity in a number of rhodophycean algae,especially Irish moss and other members of the Gigartinaceae. A crudeaqueous extract of the algae contains several carrageenans, principallykappa or lambda A) in varying proportions. The is traditionally definedas that fraction which is insoluble or forms a gel in the presence of astandard concentration of potassium ions, while the k remains insolution, e.g., Smith, Cook, and Neal, Arch. Biochem. Biophys. 53:192-204. 1954. Due to the properties of the fractions and differentindustrial uses, this type of separation based on the solubilities isusually a commercial necessity, and is both cumbersome and costly. 1tshouldbe noted that the preliminary studies of Chen et al. J. mar. biol.Ass. U.l(. 53: 11-16. 1973, were interpreted in terms of the traditionaldefinition of lambda and kappa carrageenan, whereas McCandless et al.,Planta 112: 201-212. 1973, isolated and characterized the carrageenansfrom the various Chondrus plants. In the latter work, the term lambdacarrageenan was defined as that polysaccharide fraction which mostclosely conforms to the definition of Dolan and Rees: Jour. Chem. Soc:3534-3539. 1965. The total K -soluble polysaccharide component mayinclude other carrageenans in addition to the defined by Dolan and Rees.Wide fluctuations in the ratios of KCl-insoluble to KCl-solublecarrageenans have usually been attributed to such factors as seasonalchanges, different habitats, or age of the plants.

The artificial culturing of certain algae have been carried out in thepast with very limited success. US. Pat. No. 3,195,271 Golueke et al.July 20, 1965 describes growing a red alga Porphyridiu m cruentum inseawater and sewage and adding alcohol to coagulate a form of crudecarrageenan. Certain microscopic algae have also been cultivated inconfined areas to obtain products other than carrageenans. Invariablythe algae have been grown in their normal complete life cycle.

Recently the life cycle of marine algae such as Chondrus crispus hasbecome more fully elaborated and understood, and the separation ofplants or parts thereof on the basis of their stage in the life cyclemay now be more easily and accurately made. Botanists will be able tomake the required separation into male, female and tetrasporic plants.Details on the life history of Chondrus crispus have recently beenpublished by Chen and McLachlan: Canadian Jour. Bot. 50: 1055-1060.1972, and information therein will facilitate the separations for thisspecies.

In accordance with this invention, we have now found that there is asignificant difference in the polysaccharide components of algal plantsat different stages of their life cycle. Thus male or female plants ofthese algae have been found to contain largely the insoluble or gellingform of polysaccharide (i.e. -carrageenan). Tetrasporic (asexual) plantswere' found to produce only the soluble, highly viscous form ofpolysaccharide, A-carrageenan. It is evident then that by separatingplants on this basis and separately propagating them, it is possible toobtain one form of polysaccharide in the almost complete absence of theother. Thus a post-harvest large scale separation procedure can bereplaced by a pre-growth small-scale plant selection.

We have further found that the segregated plants can be vegetativelypropagated and maintained at the selected stage of the life cycle.

The features of our invention include:

1. The selection of plant material suitable for the production of thepolysaccharide type desired;

2. The vegetative propagation of the segregated plant material or clonethereof;

3. The establishment of conditions for the successful cultivation infree-floating suspension of plants which are normally benthonic; and

4. The elimination of costly precipitation, centrifugation or K-fractionation of the harvested and extracted polysaccharide material.

The method according to the invention for the production of specificalgal polysaccharide from selected algal strains producing the desiredpolysaccharide comprises:

a. providing vegetative parts or spores separated into i. gametophytes,or

ii. tetrasporophytes (arising from either the tetrasporophytic orgametophytic stages of the plant life cycle),

b. vegetatively propagating the segregated plant material,

c. harvesting the plant growth, and

d. recovering the desired polysaccharides from the harvested plants.

The species currently of most interest are Chondrus crispus andGigartina stellara, but other carrageenanproducing species may be used.

The vegetative propagation may be carried out in any enclosed area wherenon-segregated algal material is absent. Enclosed coastal areas may beestablished for this purpose. We have found tanks or lined dug-outswithin easy pumping distance of the ocean to be quite satisfactory.Considerable work in vegetative propagation of Chondrus crispus undercontrolled conditions has been carried out by the Atlantic RegionalLaboratory of the National Research Council of Canada, Halifax, NovaScotia. Results indicate that tank culture of such algae is feasible.Results indicate that the seawater should be enriched withfixed-nitrogen-source materials such as ammonium or nitrate ions forreasonable growth rates and color retention. This nitrogen may beadministered intermittently (e.g., twice a week to give a concentrationof approximately 1 millimolar), or continuously to give an ambientconcentration greater than about 5 micromolar. Ammonium nitrate, calciumnitrate and ammonium sulfate were about equally effective in promotinggrowth. The addition of carbon as CO or NaHCO stimulated growth infertilized and in unfertilized seawater. Temperatures between l22Cpermit substantial growth of the alga, but l20C is preferred. Additionof phosphate ions appears beneficial in amounts of about one-tenth thatof the nitrogen. Growth was affected by pH and was best in the range ofpH 6.5 to 8.5. The growth medium tends to become alkaline andmaintaining the pH with CO additions was found beneficial to growthrate. A CO -maintained pH of about 7.8 was found most efficient.

It was found desirable to renew the seawater by continuous flushing toprevent a decrease of natural nutritive constituents or a build-up ofundesired products or debris. Most beneficial was the replacement of theseawater over 12 to 24 hour intervals. A low turnover of seawater (twicea week) gave cloudy water and low growth rates. Supplementalillumination favored vegetative growth compared to the natural daylightcycle. Growth was not inhibited by the absence of a dark period. Thedensity of plant material in the culture tanks is suitably within about0.2 to 1.4 lbs./sq. ft. of surface area, preferably about 0.6 1.0.Depending on the amount of illumination, plant density and agitation,growth rates at depths of up to about 6 ft. would be acceptable.

Plants growing rapidly in N-enriched seawater were found to have a lowcontent of total solids and carrageenans, and a high content ofnitrogenous compounds compared to plants growing under similarconditions in unenriched seawater. However, this solids content isreversible and in unenriched seawater the plants with a high initialN-content quickly increase in carrageenan and total solids content anddecrease in nitrogen compounds. This treatment with normal or N-depletedseawater thus becomes a desirable step before harvest. If suitableillumination is also present during this preharvest step, the plantsolids will bleach to a pale or substantially colorless state whichgives a desirable uncolored product. The duration of this pre-harveststep is suitably approximately 2 3 weeks.

This segregation and vegetative propagation technique permits theselection of starting plants or inoculum having the most vigorous growthcharacteristics. Studies at the Atlantic Regional Laboratory, N. R. C.on one plant which grew particularly well (designated T4) have shownthat a clone propagated from this plant grew from 4.5 gm. to a totalbiomass of about threefourths ton in about 28 months. Under constantconditions new growing tips were formed continually by dichotomousbranching, and when this plant was divided, the individualpieces'continued to grow well. This plant produced -carrageenan andappeared to be gametophytic.

Further experiments have shown that tetrasporic plants of Chondruscrispus increased in biomass threeto four-fold in 1 month.

The following Examples are illustrative. In some cases sporelings ofknown nuclear phases, obtained after 4 months of incubation underlaboratory conditions, were transferred to our marine laboratory. Herethey were incubated under reduced illumination (about 50% of theincident illumination in the greenhouse) in running seawater enrichedwith NH NO and (NH HPO Plants from nature were taken at Cape dOr,Cumberland Co., and Sandy Cove and Martinique Beach, Halifax Co., NovaScotia. Specimens of Gigartina stellata, derived from carpospores, wereobtained by laboratory culture under conditions as described forChondrus crispus. Plants from nature were taken at Cape dOr, Sandy Cove,Digby Co., and Sandy Cove, Halifax Co.

Before analysis, plants from nature were cleaned of all major epiphyticcontaminants. The fresh weight was recorded after blotting with papertowelling, and the plants were dried for 24 h. in the laboratory atambient temperature, then for 16 20 h. in a vacuum desiccator over CaCland finally powdered. The traditional and A-fractions were determinedcolorimetrically after extraction in aqueous sodium bicarbonate bufferand precipitation with cetyl pyridinium chloride. The precipitate wasredissolved and analysed by the color reactions obtained withresorcinol-HCl and phenolsulphuric acid.

EXAMPLE 1 Chondrus crispus from Nature (Table 1) These plants wereobtained from two widely different habitats, one on the Atlantic coast(Sandy Cove and Martinique Beach) and the other on the Bay of Fundy(Cape dOr). There was little variation in the total content ofcarrageenan among these specimens. A very marked difference in theproportions of and A-fractions was noted with the highest concentrationof the former found in the female gametophytes from both shores.Tetrasporophytes from both areas contained proportionally much moreA-carrageenan. Unfortunately no male plants were located at Cape dOr atthis collection date.

EXAMPLE 2 Chondrus crispus from laboratory culture (Tables 2 and 3)These results substantiate those obtained for plants from nature. In allcases the /)t ratio in tetrasporic plants was less than unity, and amuch higher proportion of the -fraction was found in the gametophytes.Various conditions of light, temperature and reproductive maturity hadlittle apparent effect on these ratios. This is especially evident fromthe results presented in Table 3 for tetrasporic plants.

EXAMPLE 3 Chondrus crispus from greenhouse culture (Table 4) As beforethere was a marked difference in the proportion of the and A-carrageenanin gametophytic and tetrasporic plants. Also as noted previously,reproductive maturity had no apparent effect on the IA ratio.

EXAMPLE 4 Gigartina stellata (Table 5) In plants both from nature andculture, (all included here were non-tetrasporic or gametophytic) the-fraction exceeded that of the h-fraction, and with a [A ratio similarto that in gametophytic plants of Chondrus crispus. Variation in totalcarrageenan content in these plants was not marked, and similar to thatfound in Chondrus crispus.

The isolation and characterization of the carrageenans from tetrasporicand gametophytic Chondrus wasv achieved by McCandless et al. (1973)cited above. It

was shown that the KCl-soluble carrageenans from' these two types ofplants were not identical, and that )i-carrageenan was produced only bythe tetrasporic plants while -carrageenan was formed only bygametophytic plants (e.g. FIGS. 2a-e, and 3a-d, McCandless before orafter alkali modification of the carrageenan. ln contrast to thetetrasporic plants, the bulk of the can rageenan from gametophytes wasinsoluble in KC] solution (Table 6). Sequential fractionation yielded a0.15

et al. 1973). Up to 25% of the carrageenan of gameto- 5 M KCl-insolublefraction, an intermediate fraction prephytic plants is soluble in KClsolutions and consists of cipitating between 0.15 and 1.0 M KCl and afraction the supposed precursor of -carrageenan, or mlL- soluble in 1.0M KC]. The insoluble fraction was identicarrageenan, and an unidentifiedcarrageenan. An alfied as -carrageenan by its sulfate and 3,6- kalineextraction such as is employed industrially will anhydrogalactosecontents (e.g., Table 2, McCandless convert my to -carrageenan,resulting in a product 10 et al., 1973), and an infrared spectrumindistinguishwhich is approximately 90% The carrageenan isoable fromthat of authentic -carrageenan, both deuterlated directly fromtetrasporic Chondrus is A with no ated and undeuterated. The spectrum ofthe intermediother detectable carrageenan components. ate fractionindicated that it was a mixture of -carrageenan and the 1.0 MKCl-soluble fraction (e.g. FIG. EXAMPLE 5 (Table 6) l5 2c,d,e,McCandless et al., 1973).

The chemical similarity of the carrageenans ex- Chond' 145 1" Staekhousewas Collected from the tracted from a given stage of Choridrusregardless of the intertidal Zone at Chebogue i Yarmouth County, time orplace of its collection was striking. All female Morris Point HalifaxCounty, and from 3 4 dePth plants contained -carrageenan and solublecarrageenheal' Tohey River, Pietou y in Nova seotlaonly ans other thanA-carrageenan. Extracts of these plants PldhtS bearing mature Sporahglawere used in Order to could not be distinguished from similar fractionsfrom distinguish the tetrasporophytes from the female a male plant. Onthe other hand, when extracts of the p The Single male Plant was afertile third tetrasporophyte stage were examined, only aA-carrageneration specimen maintained in culture. geenan was f d Wholecarrageenan was fractionated with KCl either These discoveries provideboth a method f h according to Smith et Arch- Biochem- Biophys- 531duction of )ior -carrageenan, and an explanation for 1954, or y amodification of the leaching the observed variability in the traditional/)t ratios. procedure of Stancioff and Stanley, Proc. Int. Seaweed E lirker did not separate the morphologically Symp. 6: 595-609- 1969- Th a gan as pr ipisimilar, but chemically different, sporophytic and gatatedby adding the solutions to 2.5 volumes of 2- 3 metophytic plants. Indeedit is remarkable that the spopropanol. The samples were washed with 80%alcohol rophytes and gametophytes should retain such a close untilchloride-free, and were dried. physical resemblance when theirstructural macromol- The data (Table 6) shows that carrageenan fromtetecules, )tor -carrageenan, exhibit very different visrasporophytesconsisted almost entirely of KCl-soluble cosities, gelling propertiesand molecular structure polysaccharide when fractionated according toSmith 35 after isolation from the plants. et al. 1954). The infraredspectra of this material, both For some purposes specific blends of thepolysacchadeuterated and undeuterated, were indistinguishable rides,different from the natural mixture, are desirable. from spectra ofauthentic A-carrageenan (e.g. FIG. By separate propagation of thedifferent types of algae 2a-e, McCandless et al., 1973 and separaterecovery of the different polysaccharides,

No -like fraction was recovered from KCl-treated various specific blendscan readily be produced from extracts of Cliondrus crispustetrasporophytes, either the products.

TABLE 1 Chandra;- Cris ms from nature carrageenan content as Date andFresh wt 7: Dry M location gms matter Total Kappa Lambda A Note February319 26.5 16.9 0.3 16.6 0.02 Tetrasporic plants Atlantic (emptysporangia) coast 3.54 i 26.8 15.0 ll.l 3.9 2.9 Female plants(carposporangia, some release of spores) L08 22.5 14.0 8.9 5.] 1.7 Maleplants March 2.ll 29.4 l5.3 2.2 l3.l 0.l7 Tctrasporic plants Bay of 2.8327.7 l5.9 l.l 14.4 0.08 Tetrasporic plants Fundy 3.60 25.l l4.6 l0.2 4.42.32 Female plants TABLE 2 Chondrus crispus from laboratory cultureCarrageenan content as Fresh wt 71 dry 71 of fresh weight gms matterTotal Kappa Lambda I) Note lncubated at 15. 16 h light period. intensity300-600 ft-c 0.78 28.0 I 1.6 8.7 2.9 2.9 Female plants (with sori) 0.9327.9 13.8 9.2 4.6 2.0 Male plants (with spormatia) 1.39 28.9 13.6 1.512.1 0.12 Tctrasporic plants (with sori) lneuhated at 13. 10 h lightperiod. intensity 300-600 ft-c 1.08 21.8 1 1.2 9.0 2.2 4.1 Female plants(with proearps; in culture 2% yrs) 0.74 23.8 12.6 1.0 1 1.6 0.09Tetrasporic plants (with sori) 1.07 27.9 16.5 1.1 15.4 0.07 Tetrasporicplants (sterile) TABLE 3 Clmndrus crispus from laboratory cultureTetrasporic plants incubated under various temperatures and photoperiodsintensity 300-600 ft-c C arrageenan content as Fresh wt 71 dry 72 offresh weight gms matter Total Kappa Lambda /A Note (plants fertile)(plants sterile) (plants fertile) (plants sterile) (plants fertile)(plants sterile) TABLE 4 Chrmdrus c'rispus from greenhouse cultureInitiated in laboratory culture and later transferred to greenhouseculture. Harvested for analyses in January.

Carrageenan content as Fresh wt. '71 dry 7: of fresh weight gms matterTotal Kappa Lambda /A Note 3.14 23.5 12.1 9.3 2.8 3.3 Female plants(fertile) 2.41 22.9 10.1 8.0 2.1 3.8 Male plants (fertile) 3.40 22.210.7 8.3 2.4 3.4 Gametophytic plants (sterile) 3.81 22.8 10.9 1.4 9.50.15 Tetrasporic plants (fertile) 3.07 22.6 10.7 1.3 9.4 0.14Tetrasporic plants (sterrle) TABLE Gigurrina srellala from nature andfrom culture Carrageenan content as Date and Fresh wt 71 Dry 7: of freshweight location gms matter Total Kappa Lambda [A Note March 2.86 33.119.9 12.8 7.1 1.8 Mature nemathecia Sandy Cove (Hfx Co.)

March 1.39 35.7 15.7 12.5 3.2 3.91 Immature nemathecia Cape d'Or April1.66 34.6 14.3 9.5 4.8 20 Plants sterile Sandy Cove (Digby Co-l March1.12 35.9 17.8 11.1 6.7 1.66 13. :12 h culture 600 ft-c maturenemathccia (discharging spores) TABLE 6 Chondrus crispus or Gigartinastellata.

5. The method of claim 2 wherein gametophytes are Data forClwndruscrispus Carrageenan vegetatively propagated and -carrageenan isrecovered. Pl' nt Method 7 of whole carra eenan sudmplc I g 6. Themethod of claim 2 wherein tetrasponc algae KCl KC! are ve etativel ro aated and A-carra eenan is reg y P P g g soluble insoluble covered L F a217 693 7. The method of claim 1 wherein the vegetative 2. F a 16.6propagation is carried out by: 3. F a, 12.8 nd 4 F b m2 (135) 5'9 1.providing seawater at a temperature suitable for the 5. M c 24.2 67.8growth of the algae, as growth medium, 9 2%? {2:3 ii. maintaining the pHin said seawater between ap- 7b. T c 94.8 2.6 proximately pH 6.5 to 8.5by C0 addition, and 8. T c 91.9 0

F=fcmale; M=male; T=tetrasporic. Fractions prepared according to:a=Stancioff at 0.3 M KCl; a,=Stancoiff at 1.0 M KCl; b=Smith et a1.sequentially treated at 0.15 and 1.1] M KCl. Data for the preparationsoluble in 0.15 M. hut insoluble in 1.0 M KCl are given in paranthcses.The other value is for material soluble in 1.0 M KC]. c=Smith ct al.using (1.3 M KCl. nd=not determined.

2. The method of claim 1 wherein the polysaccha-' rides are carrageenansand the algal strains are marine algae known to produce carrageenans.

3. The method of claim 2 wherein the strains are selected from speciesof the following genera: Chondrus, and Gigartina.

4. The method of claim 3 wherein the species are iii. maintaining thepresence of assimilable nitrogensource material to permit vigorousvegetative growth in the seawater system until shortly beforeharvesting.

8. The method of claim 7 wherein during a short period before harvestingno additional N-souree material is supplied and the plant material issubject to strong illumination.

9. The method of claim 7 wherein fresh seawater is regularly supplied tothe growth medium.

10. The method of claim 7 wherein the concentration of nitrogen-sourcematerial is maintained by continu ously or intermittently supplyingfixed nitrogen during the vigorous growth stage and then is allowed tobecome substantially exhausted for approximately 2 3 weeks beforeharvest.

11. The method of claim 10 wherein during said 2 3 week period theplants are subject to strong illumination sufficient to substantiallydecolorize the alga resulting in an improved polysaccharide material.

12. The method of claim 1 wherein different types of polysaccharides arerecovered from (i) and (ii) in separate growth areas and blended toproduce a desired polysaccharide mixture.

3,879,890 April 29, 1975 Patent No. Dated Lawrence C. M. Chen et a1.

Inventor(s) It is certified that error appears in the above-identifiedpatent and that said Letters Patent are hereby corrected as shown below:

Insert the Greek symbol K for kappa at the following locations Column 1,lines 25 and 26; Column 2, line 9,

Column 5, line 55, Column 4-, lines 12, 27, 59, l-l, 50, 52, 59 and 60;Column 5, lines 5, 7, l0, l1 and #9; Column 6, lines 8, ll, 15, 19, 26,27 and 55;

Table l, to the heading of column 7; Table 2, to the heading of columnTable 5, to the heading of column Table to the heading of column Table5, to the heading of column Column 10, line 22.

. Signed and Scaled this third Day of February 1976 [SEAL] Arrest:

RUTH C. MASON C. MARSHALL DANN Arresting Officer Commissioner ofParenrsand Trademarks FORM P040550 (0459) USCOMM-DC 60376-P69 |.|.Sv GOVERNMENTPRINTING OFFlCEI 0

1. A METHOD FOR THE PRODUCTION OF SPECIFIC POLYSACCHARIDE FROM SELECTEDALGAL SPECIES PRODUCING THE DESIRED POLYSACCHARIDE, COMPRISING: A.PROVIDING VEGATATIVE PARTS OR SPORES SEPARATED INTO I. GAMETOPHYTES, ORII. TETRASPOROPHYTES, B. VEGETATIVELY PROPAGATING THE SEGREGATED PLANTMATERIAL, C. HARVESTING THE PLANT GROWTH, AND D. RECOVERING THE DESIREDPOLYSACCHARIDES FROM THE HARVESTED PLANTS.
 2. The method of claim 1wherein the polysaccharides are carrageenans and the algal strains aremarine algae known to produce carrageenans.
 3. The method of claim 2wherein the strains are selected from species of the following genera:Chondrus, and Gigartina.
 4. The method of claim 3 wherein the speciesare Chondrus crispus or Gigartina stellata.
 5. The method of claim 2wherein gametophytes are vegetatively propagated and kappa -carrageenanis recovered.
 6. The method of claim 2 wherein tetrasporic algae arevegetatively propagated and lambda -carrageenan is recovered.
 7. Themethod of claim 1 wherein the vegetative propagation is carried out by:i. providing seawater at a temperature suitable for the growth of thealgae, as growth medium, ii. maintaining the pH in said seawater betweenapproximately pH 6.5 to 8.5 by CO2 addition, and iii. maintaining thepresence of assimilable nitrogen-source material to permit vigorousvegetative growth in the seawater system until shortly beforeharvesting.
 8. The method of claim 7 wherein during a short periodbefore harvesting no additional N-source material is supplied and theplant material is subject to strong illumination.
 9. The method of claim7 wherein fresh seawater is regularly supplied to the growth medium. 10.The method of claim 7 wherein the concentration of nitrogen-sourcematerial is maintained by continuously or intermittently supplying fixednitrogen during the vigorous growth stage and then is allowed to becomesubstantially exhausted for approximately 2 - 3 weeks before harvest.11. The method of claim 10 wherein during said 2 - 3 week period theplants are subject to strong illumination sufficient to substantiallydecolorize the alga resulting in an improved polysaccharide material.12. The method of claim 1 wherein different types of polysaccharides arerecovered from (i) and (ii) in separate growth areas and blended toproduce a desired polysaccharide mixture.